1. Field of the Invention
This invention relates to a monoclonal antibody utilized in an assay of a human growth hormone with a molecular weight of about 20,000; a cell line capable of producing the monoclonal antibody; and a sensitive immunoassay of a human growth hormone with a molecular weight of about 20,000 by means of the monoclonal antibody.
2. Description of the Related Art
It is known that in a human body, there are two main human growth hormones (sometimes referred to as "hGH"); peptide hormones with a molecular weight of about 22,000 (referred to as "22 k hGH") and about 20,000 (referred to as "20 k hGH"). 22 k hGH is a protein with 191 amino acids, while 20 k hGH is a protein with 176 amino acids.
Both 20 k hGH and 22 k hGH are proteins originally encoded in a single gene, and are, therefore, very similar in their amino acid sequences, except for a difference that 20 k hGH has a structure with a deletion of 15 amino acids of 32.sup.nd to 46.sup.th from the N-terminus of 22 k hGH.
It is known that in an adult with normal secretion, a hGH level is below about 5 ng/mL as a baseline level iin the morning.
The previous studies have shown that in a human body 22 k hGH exists much more than 20 k hGH; specifically 20 k hGH could account for 5 to 20% of the total hGHs.
The blood level of the hGHs varies constantly and largely, i.e., significantly varies within a day and increases during exercise or sleeping. However, the above findings for the quantitative ratio between 20 k hGH and 22 k hGH have been obtained by measuring hGHs extracted from a human pituitary. Therefore, the quantitative ratio between them in their natural secretion dynamics in a human body is still unknown and whether it varies in a short time is also unknown.
As a drug, 22 k hGH has been marketed, and has been studied more intensively than 20 k hGH.
The biological activities of 20 k hGH have been recently reported; its growth-promoting activity is comparable to that of 22 k hGH, is less inducible in an abnormal glucose tolerance and could be less diabetogenous (Lewis, U. J., et al., Endocriniol. Japon, 34, 73-85(1987)). This suggests that when a human growth hormone is used as a drug 20 k hGH could have less risk of adverse drug reactions than 22 k hGH. Thus, usefulness of 20 k hGH has been increasingly paid much attention as a drug for a substitution therapy to an adult with growth hormone deficiency (Hisao Seno, Igaku No Ayumi, 165, 247-251(1993)).
However, there had been no successful cases for producing a large amount of pure 20 k hGH before the present inventors have first established a preparation process recently, and thus there has not been obtained 20 k hGH with sufficient amount and purity to conduct a variety of studies. Therefore, 20 k hGH has never been clinically applied. In addition, since there have been not developed a sensitive immunoassay for 20 k hGH, it has been difficult for a long time to determine relationship between the activities and the molecular structures of 20 k hGH and 22 k hGH. The previous studies for 20 k hGH have been mainly in a molecular or cellular level using a small amount of 20 k hGH extracted from a living body, and there have been no clinical studies for 20 k hGH.
The determination methods of hGHs will be described. There are several known determination methods for hGHs in human blood or urine to explicate biological roles of hGHs (e.g., Hashida and Ishikawa et al., Clinica. Chimica. Acta, 162, 229-235(1987)).
It is essential for developing hGHs as a drug that an accurate secretion dyanamics is determined in a clinical trial, including a baseline blood level of hGHs in a human body. In addition, the determination method is required to be sensitive with no errors.
Since 22 k hGH has been clinically applied, there are immunoassays capable of determining a baseline blood level in the morning for 22 k hGH (below 5 ng/mL).
For example, for 22 k hGH, an immunoassy using an antibody specific to 22 k hGH has been disclosed in Japanese Patent Laid-Open No. 273496/88 (JP-A 63-273496). The specification describes that its cross-reactivity to 20 k hGH is 15% However, since the purity level of the standard 20 k hGH used in the method is not described, the above percentage may be quite questionable.
On the other hand, all of the previous examples for immunoassays for 20 k hGH were to measure around a higher value (1 ng/mL) of 20 k hGH secretion under exercise load. Any of the previous determination methods has a performance only for investigating human 20 k hGH in cellular level due to its inadequate cross-reactivity and sensitivity, i.e., does not have an adequate sensitivity to determine its baseline blood level.
The known immunoassays for 20 k hGH are as follows.
There is an assay wherein a value for 20 k hGH is obtained by subtracting a measured value for an antibody specifically reacting with 22 k hGH from a measured value for an antibody reacting with all hGHs (Eur. J. Appl. Physiol., 62, 130-134(1991)). The author has assumed that hGHs other than 22 k hGH would consist of 20 k hGH. Recently, several variants of hGH have been discovered, which will react with an antibody as total hGHs like 20 k hGH. Therefore, the author would probably have determined the variants as 20 k hGH, and thus the measured values for 20 k hGH must be quite inaccurate.
A monoclonal antibody specific to 20 k hGH has been disclosed by Mario Mellado, et al. (Journal of Clinica Endocrinology and Metabolism, 81, 1613-1618(1996)), who describes that cross-reactivity to 22 k hGH is below 1%, and does not demonstrate an actual example of determination of 20 k hGH in human body fluid. As discussed later, it is impossible in theory to elucidate the secretion dynamics in a human body of 20 k hGH using the monoclonal antibody of this article.
Hence, there have been no sensitive immunoassays capable of evaluating the secretion dynamics of 20 k hGH. Determining the secretion dynamics of 20 k hGH means that 20 k hGH can be accurately determined in a relatively low level near its baseline blood level. For an immunoassay of 22 k hGH, the situation is similar in terms of accurate determination in relatively lower level around its baseline blood level. For 20 k hGH, there are, however, intrinsic problems in cross-reactivity and sensitivity, but not for 22 k hGH. Thus, it has been difficult to provide a sensitive immunoassay for 20 k hGH in contrast to the immunoassays for 22 k hGH.
Cross-reactivity is a phenomenon observed when an epitope reactive to an antibody is also reactive to another antibody. The cross-reactivity should be taken into consideration in an immunoassay for 22 k hGH. However, it will not be a significant problem because of the following reasons: First, 22 k hGH is contained in blood much more than 20 k hGH (5 to 20 times). Next, as described above, 22 k hGH has a unique sequence consisting of 15 amino acids. Therefore, if a monoclonal antibody capable of recognizing the unique sequence as an epitope could be prepared, a monoclonal antibody which may specifically react with 22 k hGH and be adequately sensitive would be readily prepared.
On the other hand, for an immunoassay for 20 k hGH, it is difficult to solve the problems of cross-reactivity and sensitivity due to the following reasons.
First, 20 k hGH is much less than 22 k hGH in blood (1/5 to 1/20). This simply means that a sensitive immunoassay for 20 k hGH is required to has a sensitivity 10 times higher than a sensitive immunoassay for 22 k hGH. Furthermore, cross-reactivity to 22 k hGH significantly affects the precision in the measurement.
Next, 20 k hGH is different from 22 k hGH in that it does not have a unique amino acid sequence. If anything, the ligation segment between the termini of the deletion of the 15 amino acids from 22 k hGH, i.e., the sequence wherein 31.sup.st phenylalanine and 32.sup.nd asparagine (47.sup.th amino acid from N-terminus in 22 k hGH) from N-terminus are adjacent to each other, might be considered to be a unique amino acid sequence in 20 k hGH. Lewis et al have immunized an animal with a polypeptide coupled with albumin containing a region in the vicinity of the amino acid sequence (an amino acid sequence of 28.sup.th to 38.sup.th from N-terminus of 20 k hGH) and attempted to prepare a monoclonal antibody recognizing the polypeptide as an epitope, but have been unsuccessful (Lewis U. J. et al, Endocrinol Japon, Vol.34, pp.73-85(1987)).
Thirdly, to prepare a monoclonal antibody which is little cross-reactive to 22 k hGH, but is adequately sensitive, it is necessary to separate a number of cells producing a monoclonal antibody, from which the cells producing the desired monoclonal antibody are then selected. Furthermore, in this case, the animals for preparing the antibody-producing cells should be adequately immunized, requiring a large amount of pure 20 k hGH.
To date 20 k hGH has not been commercially available and there are few methods for its preparation. In such a situation, it is difficult to obtain 20 k hGH in an amount to adequately immunize an animal. Before this invention, it has been difficult to adequately immunize an animal with 20 k hGH for preparing antibody-producing cells. Consequently, it has been quite difficult to obtain cells producing the desired monoclonal antibody.
Thus, in contrast to a monoclonal antibody specific to 22 k hGH, it has been hard to obtain a monoclonal antibody specific to 20 k hGH, and therefore, it has been difficult to establish a sensitive immunoassay for 20 k hGH using the monoclonal antibody.
The followings will describe the problems of cross-reactivity and sensitivity in a sensitive immunoassay for 20 k hGH in terms of the above process described by Mellado et al.
Mellado et al. describe that the monoclonal antibody reactive to 20 k hGH has below 1% of cross-reactivity to 22 k hGH. If a sample containing 5 ng/mL of 22 k hGH and 250 pg/mL of 20 k hGH is subject to an immunoassay using a monoclonal antibody with 1% of cross-reactivity, a measured value will be 5000 pg/mL.times.0.01+250 pg/mL=300 pg/mL, giving an error of 20% compared with the true value, which indicates that determination of a baseline blood level is not feasible.
In addition, Mellado et al. describe that the measuring sensitivity of the immunoassay is 4 ng/mL. Since a peak level of 20 k hGH in human blood is about 1 ng/mL for a normal adult, the immunoassay described by Mellado et al. can never determine the baseline blood level of 20 k hGH. In other words, this method can never elucidate the secretion dynamics of 20 k hGH in human blood.
As described above, Mellado et al. have not solved the above problems of cross-reactivity and sensitivity in an immunoassay for 20 k hGH. That is, the monoclonal antibody described by Mellado et al. and the immunoassay therewith cannot be applied to a clinical study for development of 20 k hGH as a drug.